Soil hosts most of the biodiversity in the environment, where each cubic centimeter of soil matrix can contain hundreds of thousands of microorganisms that cohabitate in a complex assemblage of mineral and organic matter. The structure and function of microbial communities are dynamic processes that play important and beneficial roles in productivity of ecosystems, including oxygen production, crop growth, bioremediation, carbon sequestration, nitrogen fixation, and water purification. Simultaneously, microbial species may act as pathogens for living organisms. For example, plants from hundreds of different species are killed annually in Australia by P. cinnamomi; and grain development, in wheat, is affected by infection of G. graminis var. tritici in vascular tissue. Therefore, there is a need to develop the probes and assays that enable studying microbial species in their native environment, i.e., in situ imaging. Applications of in situ imaging include, but are not limited to, the insights and understanding of the (i) composition and population of a normal gut microbiome as a function of exposure to antibiotics and/or under environmental stress; (ii) interactions and cross talk between microbes and plant roots in rhizosphere; (iii) localization of endophytes in healthy plant tissues for improved yield; and (iv) profiling of the microbial communities in soil crust for erosion control, water retention, and nutrient cycling.
To meet the requirements of in situ imaging and identification of microorganisms, synthesized probes must (i) penetrate the cell wall and lipid membrane, (ii) be non-sticky to the soil matrix, and (iii) differentiate between living and dead microorganisms. Previously, guanidinium-rich molecular transporters (GR-MoTrs) have been demonstrated to be internalized in different strains of algae by crossing both the cell wall and the lipid membrane; however, it was later discovered that these molecular transporters were sticky to the matrix substrate. Other polymer-based nanoparticles, such as lipofectamine, have also been found to be sticky to the natural environment. Moreover, in some cases, synthesized probes ideally should facilitate radiolabeling to meet the general requirements of in situ imaging. For example, the structure of a microbial community can be imaged with x-ray microtomography and MRI, but these techniques are destructive and do not report biological activities, the successful imaging of which is highly dependent on the design of the imaging instruments. Thus, a need still exists to develop probes that allow efficient in situ visualization of microbial density that overcome the problems associated with the currently available technologies.
There is thus a need in the art for probes that allow efficient in situ visualization of microbial presence and density that are non-destructive and non-invasive. Additionally, there remains a need in the art for probes and methods which are capable of differentiating microbial populations quickly, cheaply and effectively. The present invention fulfills these needs.